Much evidence has indicated that a number of protein kinases act to control cell division in eukaryotic cells. A variety of oncogenes encode cellular protein kinases. The gene products of mammalian ras genes are not protein kinases, but they do function in yeast (Saccharomyces cerevisiae) to activate adenylate cyclase; the intracellular cAMP in turn activates a protein kinase. Why is cAMP-dependent protein kinase (protein kinase A) activity essential for the viability of S. cerevisiae and what metabolic processes are controlled by cAMP concentration in this organism? The major intracellular receptor for cAMP is the regulatory subunit of protein kinase A, encoded by the SRAl (BCYl) gene. Mutations in SRAl suppress RAS and confer a variety of pleiotropic phenotypes. Biochemical experiments are proposed to critically test a hypothesis about the nature of the sral mutations and explain the variety of phenotypes. Specifically, mutant regulatory proteins purified from yeast will be assayed for cAMP binding, interaction with catalytic subunit, phosphorylation in vitro and in vivo, and subcellular localization. The DNA sequence alterations of mutant SRAl genes will allow delineation of functional domains to be made for this protein. The traits displayed by sral mutants are probably due to constitutive or over phosphorylation of protein kinase A substrates. The reversion of these traits provides an avenue to the identification of protein kinase A substrates and phosphoprotein phosphatases because they will be the primary extragenic suppressors of SRA1. What are the functions of protein kinase A substrates and how are they modulated by phosphorylation? The answers to these questions will come from a thorough characterization of SRA1 suppressor genes. This analysis will minimally entail (in chronological order) phenotypic characterization, genetic mapping, gene cloning, DNA sequencing, and protein homology searches. These same techniques (beginning at the cloning step) will be applied to determine the function of the SRA7 gene; mutants in which suppress RAS but fail to show traits exhibited by other RAS suppressors.
